1. Field of the Invention
This invention relates to a process for the preparation of D-pantothenic acid and/or a salt thereof using a microorganism of the Enterobacteriaceae family in which at least the poxB gene is attenuated.
2. Discussion of the Background
Pantothenic acid is produced worldwide in an order of magnitude of several thousand tons a year. It is used, inter alia, in human medicine, in the pharmaceuticals industry and in the foodstuffs industry. A large portion of the pantothenic acid produced is used for nutrition of stock animals such as poultry and pigs.
Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by fermentation of suitable microorganisms in suitable nutrient solutions. In the chemical synthesis, DL-pantolactone is an important precursor. It is prepared in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide. In further process steps, the racemic mixture is separated, D-pantolactone is subjected to a condensation reaction with xcex2-alanine, and D-pantothenic acid is obtained.
The typical commercial form is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture of D,L-pantothenic acid is also customary.
The advantage of the fermentative preparation by microorganisms lies in the direct formation of the desired stereoisomeric D-form, which is free from L-pantothenic acid.
Various types of bacteria, such as Escherichia coli (E. coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, and also yeasts, such as Debaromyces castellii, can produce D-pantothenic acid in a nutrient solution which comprises glucose, DL-pantoic acid and xcex2-alanine, as shown in EP-A 0 493 060. This patent furthermore shows that in the case of E. coli the formation of D-pantothenic acid is improved by amplification of pantothenic acid biosynthesis genes from E. coli which are contained on the plasmids pFV3 and pFV5 in a nutrient solution comprising glucose, DL-pantoic acid and xcex2-alanine.
EP-A 0 590 857 and U.S. Pat. No. 5,518,906 describe mutants derived from E. coli strain IF03547, such as FV5714, FV525, FV814, FV521, FV221, FV6051 and FV5069, which carry resistances to various antimetabolites, such as salicylic acid, xcex1-ketobutyric acid, xcex2-hydroxyaspartic acid, o-methylthreonine and xcex1-ketoisovaleric acid. They produce pantoic acid in a nutrient solution comprising glucose, and D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine. It is furthermore stated in EP-A 0 590 857 and U.S. Pat. No. 5,518,906 that after amplification of the pantothenic acid biosynthesis genes panB, panC and panD, which are said to be contained on the plasmid pFV31, in the above-mentioned strains the production of D-pantoic acid in nutrient solutions comprising glucose and the production of D-pantothenic acid in a nutrient solution comprising glucose and xcex2-alanine is improved.
The favorable effect of enhancement of the ilvGM operon on production of D-pantothenic acid is furthermore reported in WO97/10340. Finally, the effect of enhancement of the panE gene on the formation of D-pantothenic acid is reported in EP-A-1001027.
According to the prior art, D-pantothenic acid or the corresponding salt is isolated from the fermentation broth and purified (EP-A-0590857 and WO96/33283) and accordingly used in purified form, or the fermentation broth comprising D-pantothenic acid is dried in total (EP-A-1050219) and used in particular as a feedstuffs additive.
It is an object of the present invention to provide a novel method for the preparation of D-pantothenic acid and/or a salt thereof.
It is another object of the present invention to provide an animal feedstuffs additive comprising D-pantothenic acid and/or its salt.
These and other objects have been achieved by the present invention the first embodiment which includes a process for the preparation of D-pantothenic acid and/or a salt thereof comprising:
preparing D-pantothenic acid and/or a salt thereof by fermentation of a microorganism of the Enterobacteriaceae family in which at least a nucleotide sequence which codes for a poxB gene is attenuated, thereby attenuating an intracellular activity of pyruvate oxidase.